Project 3. Immune Analysis of Clinical Trial Samples. Abstract The reasons for the lack of therapeutic success of CAR therapy for solid tumors, compared to that seen in hematologic malignancies, represents one of the most important questions in the field of immunotherapy. Programs that can integrate clinical trials (Project 1), preclinical studies (Project 2) and in-depth biocorrelate studies of samples derived from these human trials (this Project) are needed. As described in Project 1, our new clinical trial will use an optimized mesothelin targeted CAR, M5 huCART-meso, that has been constructed to contain a new anti-mesothelin scFv fragment that it is fully human (selected from a phage display library). M5 huCART-meso also had better preclinical efficacy than our previous SS1-mesothelin CAR (see Project 1). Our second planned clinical trial will use a CAR, huCART-FAP, directed to fibroblasts in tumor stroma by targeting fibroblast activation protein (FAP). The goal of Project 3 is to conduct biocorrelate studies of these clinical trials. We will focus on two main areas. In Aim 1, we will conduct studies to learn whether our CAR T cells in Project 1 are persisting and trafficking to tumors. We will analyze blood samples from our patients in Core B, however, we hypothesize that studying the ability of CARs to traffic to tumors is even more critical. CAR T cells isolated from tumor biopsies will be characterized for phenotype and function. In addition, the tumor will be examined for histological changes, target antigen expression, and transcriptionally profiled. Post-treatment biopsies will be expected from each patient in the trials proposed in Project 1 helping to ensure that materials from tumors are available for such studies. In Aims 2 and 3, we will conduct experiments aimed at studying epitope spreading which is a crucial, yet an understudied area of adoptive T cell transfer. In Aim 2, we will evaluate the hypothesis that the CAR T cells used in our clinical trials can enhanced/promote endogenous T cell immunity to shared tumor antigens (Aim 2A) and neoantigens (Aim 2B). In Aim 2A, overlapping peptide libraries from known tumor antigens as well as defined peptides will be used to interrogate PBMC for responses to shared tumor antigens. In Aim 2B, we will use next generation sequencing (NGS) of tumors to identify candidate neoepitopes. Tandem mini-gene constructs, as well as synthetic peptides encoding these neoepitopes, will be used to interrogate peripheral blood (and in limited cases, TIL) for new or enhanced T cell immunity after CAR therapy. Aim 2 in Project 3 is linked to Aim 2 in Project 2, where investigators will use animal models to look for the presence of antigen spreading and ways to augment this effect. Finally, in Aim 3 we will examine the hypothesis that CAR-mediated epitope spreading promotes a diverse repertoire of antigen-specific T cell responses in tumors by utilizing NSG methods to characterize TCR clonotypes in shared/neoantigen-specific T cell lines derived from blood. These TCR reference libraries will be employed to interrogate pre- and post- CAR T therapy in blood/tumor biopsy samples in order to characterize the intra-tumoral breath and composition of T cell immunity promoted by CAR-mediated epitope spreading.